This application is a 371 of PCT/EP00/04517 filed May 18, 2000.
The present invention is related to a method for providing a substrate structure for oriented neurite outgrowth as well as to the respective substrate structure itself. The present invention is further related to a device for monitoring cell or neuron activity, comprising a substrate, at least one electronic device and at least one neuron or cell coupled with said electronic device.
During the past decades there has been a growing interest in the development of experimental systems, in which a small number of neurons are grown in defined patterns. It is a goal to provide a neuro-electronic system to investigate the structure-function relation of neural architectures.
Individual neurons can be stimulated and the electric activity and/or changes in membrane potential of neurons can be recorded and investigated.
The technological products and outputs arising from such investigations and respective devices will be especially the design of test systems for pharmaceutical development, prosthetic devices and neural network engineering.
Very important for the above-mentioned products and investigations is the physical interfacing, i.e. an extremely close contact of the nerve cells or neurons with the artificial substrate, especially to achieve the desired coupling between individual neurons and e.g. respective monitoring devices.
Regarding the physical interfacing, interactions of cultured neurons with defined surfaces are of a very high importance. The control of the function and geometric patterns of neurons in vitro, i.e. in an artificial environment, is dependent on the interaction of the cells with the culture substrate.
In order to avoid biosystems to denature in contact with the artificial substrate surfaces it is known to first treat substrate surfaces with biological relevant substances to improve neuronal survival. Peptide polymers, e.g. polylysine (PL) with pendent ε-amine groups are suggested as substrate materials for many types of neurons in culture, see e.g. “Interfacing Neurons and Silicon by Electrical Induction”, P. Fromherz, Ber. Bunsenges. Phys. Chem. 1996, 100, 1093–1102.
It is further known to use self-assembled monolayers to create artificial surfaces for in vitro neuronal culture. Chemical functional groups can be incorporated into silane or thiol molecules used for self-assemby, see book of D. Stenger and T. McKenna, “Enabling Technologies for Cultured Neural Networks”, Academic Press, Inc., 1994, Chapter 4.
To study interactions of neurons with their environment and with other neurons it is extremely important to control the adhesion and the geometry of the neurite outgrowth. It is therefore necessary to prepare surfaces with well-resolved areas to control the neurite outgrowth. In this respect, it is known to use lithographic technologies to structure the artificial surfaces of the substrate to allow chemical groups to be spatially patterned. In this connection e.g. photoresist, photopolymerisation, or deep UV light are used within the lithographic technologies.
For monitoring electrical activity of neurons, it is known to use intra-cellular methods, e.g. using glass microelectrodes or patch clamp pipettes. Although such devices can be cheaply produced, they are, as being intra-cellular devices as mentioned above, causing damages to the neurons or cells and therefore are not capable for long-term recording. Furthermore, investigation of complex systems is very difficult with these methods.
It is also known to use extra-cellular electrodes for monitoring electrical activities, e.g. by using microelectrodes or field effect transistors (FET), as described in a paper of Offenhäusser et al. “Field-Effect transistor array for monitoring neurons in culture”. Biosensors and Bioelectronics 1997, Volume 12, No. 8, 819–826.
It is also known to use voltage sensitive dyes as optical probes for monitoring optical parameters directly related to neural activity, as described by Parsons et al., Biophys. Journal 1989, 56, 213. Disadvantages of the dyes are the toxicity in illumination, which also makes them unsuitable for long-term applications. Furthermore, stimulation of neurons using this method is not possible.
For an efficient investigation of a neural activity and for the above-mentioned devices it is extremely important to control the growth of cultured neurons on the substrate. A method using surfaces modified with e.g. artificial polypeptides, silanes or thiols with different end groups is only suitable for limited surfaces, like silicon or noble metal surfaces. Further and even more important, the random spatial distribution and overlapping of dendrites and axons on homogeneous substrates makes geometrically dependent studies of synaptic functions impossible.
The above-described lithographic techniques for structuring the surface, whereby the neurite outgrowth is oriented along the pattern, which is normally defined by a grid, are, on the other hand, methods requiring additional preparation steps. Furthermore, the structure has to be determined before applying the lithographic techniques and well before starting the neurite outgrowth, therefore no flexibility regarding parameters and fitting to desired applications or investigations is given. Differently structured substrates will have to be hold on stock in order to have a suitable substrate always available for the desired application.
Furthermore, as the lithographic structure is normally defined by a grid, it only allows a contact between neurons to the respective next neighbors, thereby clearly limiting the number of contacts and also limiting the speed of signal processing between neurons not being next-neighbour-neurons.